Activation of ATPase activity of simian virus 40 large T antigen by the covalent affinity analog of ATP, fluorosulfonylbenzoyl 5'-adenosine.
Activation of ATPase activity of simian virus 40 large T antigen by the covalent affinity analog of ATP, fluorosulfonylbenzoyl 5'-adenosine.
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ATP 的共价亲和类似物氟磺酰基苯甲酰 5-腺苷激活猿病毒 40 大 T 抗原的 ATP 酶活性。
DOI：
10.1128/jvi.64.10.4939-4947.1990
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发表时间：
1990
影响因子：
5.4
通讯作者：
Bradley,MK
中科院分区：
文献类型：
--
作者：
Bradley,MK
氟磺酰苯甲酰5‘-腺苷(FSBA)结合在猴病毒40大T抗原(T)的一个位点上，与95%以上的分子发生完全反应。这种对ATP的类似物特别地与ATP结合位点中的磷酸镁口袋交联。用溴化氰裂解和胰酶消化[14C]FSBA标记的蛋白质，结合T特异性的单抗分析，将T上的位置定位到胰酶多肽，恰好C端定位到PAb204表位。FSBA连锁的位置与先前描述的T中ATP结合区的预测三级结构一致(M.K.Bradley，T.F.Smith，R.H.Lathrop，D.M.Livingston和T.A.Webster，Proc.娜塔莉。阿卡德。SCI。美国84：4026-4030,1987)。FSBA与T的结合是协同的，这意味着两个结合位点之间存在相互作用。这可能发生在蛋白质形成二聚体的情况下，而且已知ATPase活性与二聚体T有关。最有趣的是当高达50%的T被类似物结合时，ATPase被激活。这种效应也是通过在高温下与毫摩尔浓度的三磷酸腺苷或不可水解的类似物β-亚甲基5‘-腺苷二磷酸预孵育而产生的。当T的50%以上被FSBA修饰时，ATPase被抑制，因为类似物交叉连接到第二个先前激活的结合位点。这些数据支持T中的一个ATP结合位点作为调节和催化的双重功能。
Fluorosulfonylbenzoyl 5'-adenosine (FSBA) bound to one site in simian virus 40 large T antigen (T) and covalently modified greater than 95% of the molecules in a complete reaction. This analog for ATP specifically cross-links to the Mg-phosphate pocket in ATP-binding sites. Cyanogen bromide cleavage and tryptic digestion of [14C]FSBA-labeled protein, paired with T-specific monoclonal antibody analyses, were used to map the site in T to a tryptic peptide just C terminal to the PAb204 epitope. The location of the FSBA linkage was consistent with the predicted tertiary structure of the ATP-binding region in T described previously (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Binding of FSBA to T was cooperative, implying an interaction between two binding sites. This could occur if the protein formed a dimer, and it is known that the ATPase activity is associated with a dimeric T. Most interesting was the activation of the ATPase when up to 50% of T was bound by the analog. The effect was also produced by preincubation with millimolar concentrations of ATP or the nonhydrolyzable analog gamma beta-methylene 5'-adenosine diphosphate at elevated temperatures. When greater than 50% of T was modified by FSBA, the ATPase was inhibited as the analog cross-linked to the second, previously activated, binding site. These data support a dual function for the one ATP-binding site in T as both regulatory and catalytic.